The mechanism underlying the onset of ulcerative colitis (UC) has not yet been elucidated in detail. Unknown components in colorectal tissue may be important risk factors to elucidate the cause of UC; however, they have not been highlighted as targets. To identify key factors, rats with dextran sulfate sodium-induced experimental colitis were used. The level of carbonic anhydrase III was significantly decreased in both the serum and colon tissues of these UC rats. Upon stimulation of peritoneal macrophages (MΦ) with lipopolysaccharide, the intracellular concentration of carbonic anhydrase III significantly decreased, while the secretion of pro-inflammatory cytokines from MΦ treated with an anti-carbonic anhydrase III antibody was negatively regulated. In conclusion, carbonic anhydrase III may be a novel regulator of experimental colitis in rats.in vitro assay, CA-III function in peritoneal macrophages (MΦ) from rats was investigated. Upon stimulation of MΦ with lipopolysaccharide (LPS), the CA-III concentration significantly decreased in the cytoplasm of these cells. MΦ treated with an anti-CAIII antibody followed by stimulation with LPS actively secreted inflammatory cytokines, particularly interleukin-6 and tumor necrosis factor-α. Therefore, CA-III in MΦ appears to be an immune regulator that suppresses the secretion of inflammatory cytokines.Ulcerative colitis (UC) is characterized by chronic inflammation of the large intestine, repeated remissions, and symptom relapses. Although unknown components in colonic regions are deeply involved in the pathogenesis of UC, the causes of UC development and aggravation have not yet been elucidated in detail. To identify key factors, we investigated the changes in protein components in the large intestine of rats with dextran sulfate sodium-induced experimental colitis (UCR). The components that differed in their concentration between normal rats (WT) and UCR were carefully investigated by electrophoretic separation and mass spectrometry. Based on these results, seven proteins with different expression levels between the WT and UCR were observed. Among them, we focused on carbonic anhydrase III (CA-III) in the pathogenesis of UC. CA-III concentrations in the colon tissue and serum were quantitatively measured using an enzyme-linked immunosorbent assay (ELISA) and real-time PCR, and the levels significantly decreased in both the colon tissue and serum of UCR with the aggravation of experimental UC. In an  Ulcerative colitis (UC) is classified as an inflammatory bowel disease (IBD) and is characterized by chronic and recurrent inflammation in the large intestine, particularly in the rectal region ,2. A lar2-macrogulobulin concentrations correlated with DAI scores in these patients [The S100 proteins , the levels of which fluctuate in the large intestine, have been the focus of research on the pathogenesis of UC. They have been strongly implicated in the aggravation of UC via toll-like receptor 4 or receptor for advanced glycation end products . We prevpatients . Leucinepatients ,19. AlthMany researchers have suggested that inflammatory cytokines and CXCL chemokines are useful biomarkers in UC, with inhibition of their secretion potentially contributing to the control of inflammation ,21. ActiThe animal experiments complied with ARRIVE guidelines and were approved by the Animal Experiment Committee of Kyoto Tachibana University (permission number: 20-03).Wild type Slc:Wistar rats  were purchased from Shimizu Laboratory Supplies Co., Ltd. . They were housed for approximately one week prior to the experiments and were allowed free access to food and water.TM Protein Extraction Solution (Cell/Tissue) were purchased from Cosmo Bio Co., Ltd. . The DSS salt (MW: 36000–50000) was purchased from Wako Pure Chemical Industries, Ltd. . Clinical thioglycollate medium (E-MC17) was obtained from Eiken Chemical Co., Ltd. . LPS from Escherichia coli was purchased from Sigma-Aldrich Co., LLC . VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Vector Inc. . N-Hydroxysuccinimidobiotin (EZ-LinkTM NHS-Biotin), TRIzolTM reagent, SuperScriptTM II Reverse Transcriptase, PowerUp SYBR Green Master Mix, and all primers were purchased from Thermo Fisher Scientific . All other reagents were obtained from Wakenyaku Co., Ltd. , Nacalai Tesque Co., Ltd. , and Bio-Rad Laboratories, Inc. .Pre-packed disposable PD-10 columns were purchased from Global Life Science Technologies Japan Co., Ltd. . Anti-CA3/carbonic anhydrase III mouse monoclonal antibody (anti-CAIII Ab) was obtained from Funakoshi Co., Ltd. . Anti-mouse IgG (goat)-horseradish peroxidase (HRP) or -fluorescein 5-isothiocyanate (FITC) conjugate and streptavidin (STA)-tetramethylrhodamine (TRITC) were purchased from Abcam . The CA3/Carbonic anhydrase III enzyme-linked immunosorbent assay (ELISA) kit and PRO-PREPTo generate experimental UC model rats , WT rats were orally administered 5% DSS in distilled water (DW) for 10 days. During this period, the body weight of each rat was measured every morning, and the DAI scores were evaluated  based onTM Protein Extraction Solution (Cell/Tissue) for 10 min and then centrifuged at 12,000 rpm at 4 °C for 10 min. The resultant supernatants were transferred into 1.5 mL polycarbonate tubes and stored at −80 °C until use.The three tissue segments  collected from each rat were independently weighed. Three hundred micrograms of each sample was incubated in 0.5 mL PRO-PREPTM reagent for 10 min and then centrifuged at 12,000 rpm at 4 °C for 15 min. The mRNA from each sample was extracted according to the manufacturer’s protocol and then treated with 8 M LiCl to avoid the influence of DSS on RNA reverse transcription. cDNA was synthesized from the mRNA using SuperScriptTM II Reverse Transcriptase, as described in the instruction manual.Three hundred micrograms of the three tissue segments was independently incubated in 1.0 mL TRIzolProtein extracts collected from the rectum of the UCR on Day 10  were mixed in a 1.5 mL polycarbonate tube. The mixture of protein extracts was subsequently added to a PD-10 column for GFC. The column was thoroughly washed with 0.3 mL of 10 mM Tris-HCl buffer (pH 7.4) and 0.9% NaCl (buffer A), and 0.3 mL samples of each wash fraction were collected in microtubes (1.5 mL). This procedure was repeated until 18 fractions were collected in the microtubes. The same experiment was conducted using protein extracts from the rectum of WT rats (n = 5) as a negative control.After GFC, the proteins in each fraction were separated by SDS-PAGE in the presence of 2-mercaptoethanol, as previously described . The conAfter SDS-PAGE, some protein bands were excised and delivered to the MALDI-TOF mass spectrometry accession service  for analyses. Briefly, a single protein band was digested with trypsin, and peptide mass fingerprint analysis was conducted using Microflex LRF 20 . The fragments of protein components were identified using a Mascot search of the National Center for Biotechnology Information (NCBI) database. Mascot scores >67 were considered statistically significant.Carbonic anhydrase III (CA-III) in each sample was measured using an ELISA kit, according to the manufacturer’s instructions. The absorbance of the color reaction was measured at 450 nm using a microplate reader .We microscopically observed rectal tissues with severe inflammation in the DSS-induced UC rats. Three micrometer thick tissue sections obtained from the rectal tissues of UCR and WT rats were stained with hematoxylin and eosin (H&E). Tissue damage was histologically evaluated and assessed based on the H&E staining, and the extent of damage was scored based on the criteria shown in TM XRS Plus Imaging System and Clarity Western ECL substrate .After SDS-PAGE, the proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo . After the membranes were blocked with Blocking One , they were incubated at 4 °C for 1 h with 2 μg/mL of anti-CAIII Ab. The membranes were then washed three times for 5 min with buffer A, twice with buffer A/0.1% Tween 20, and once with buffer A before being incubated with 2 μg/mL rabbit anti-mouse IgG H&L (HRP) at room temperature for 1 h. After the membranes were washed, antibody-bound proteins were detected using the ChemiDocReal-time PCR was performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) as previously described . The pri4Cl and incubated at 37 °C for 10 min to induce hemolysis of the contaminating erythrocytes. After centrifugation as above, the supernatant was discarded, and the pelleted cells were suspended in RPMI-1640 culture medium containing 10% fetal bovine serum . A total of 2 × 106 cells were plated in each well of a six well plate, and an appropriate volume of medium A was added. The cells were then incubated at 37 °C for 2 h in 5% CO2. After incubation, the non-adherent cells in each well were removed by washing three times with buffer B. The adherent cells were maintained in the same medium at 37 °C in 5% CO2 until use.MΦ were isolated from WT rats as previously described . BrieflyTM Protein Extraction Solution and TRIzol™ reagent, respectively. The intracellular expression of CA-III was evaluated using western blotting, real-time PCR, and FICS.After thorough washing with buffer B, the MΦ were stimulated with LPS (1 μg/mL) in medium A for 0.5, 1, or 2 h. After washing three times with buffer B, the cells were subjected to FICS or collected with 0.2 mL of buffer A in a polycarbonate tube (1.5 mL). Protein and mRNA were extracted from the collected cells using PRO-PREPp < 0.05. The relationship between the two groups in each case was assessed by Spearman’s test using the statistical software “Easy R version 1.54” [Pairwise comparisons with controls were performed using non-parametric tests. Significant differences between the groups were identified using the Mann–Whitney U test. Data are shown as the mean ± standard deviation (SD). Statistical significance was set at on 1.54” . A correH&E staining was performed to visualize the tissue damage and immune cells in the rectal region. Comparisons with WT tissue revealed that the epithelial structure of the rectal region of the UCR was damaged, and a large number of immune cells had infiltrated the tissue during the course of the experimental period A. We evaProteins in the rectal region of WT and UCR were fractionated by GFC according to our original method see . After sApart from a few protein bands , six residual protein bands yielded good scores (>67). Consequently, seven proteins  were identified based on information available from the NCBI database . The resTo observe differences in the extent of CA-III protein expression in the rectal region of the large colon of UCR, immunohistochemical staining was performed using a specific antibody for CA-III, in which DAB was used as a substrate for color development. CA-III was mainly expressed in the normal mucosal epithelium in the rectal tissue of WT, whereas the CA-III-positive region gradually shrunk in size and ultimately disappeared with the aggravation of UC A. ELISA ELISA, real-time PCR, and Western blotting were performed to investigate the expression of CA-III in more detail. ELISA showed that CA-III in the three colon fragments gradually decreased over the course of the experimental period A. In addThroughout the course of the in vivo study, we speculated that CA-III plays a pivotal role in MΦ in the large colon tissues of rats with experimental UC. An in vitro study was conducted to test our hypothesis using MΦ derived from WT. The amount of CA-III in the MΦ gradually decreased after LPS stimulation . Real-tiWestern blotting showed that the expression of CA-III decreased to some extent in MΦ treated with anti-CAIII Ab . FurtherFive cytokines in active MΦ treated with/without anti-CAIII were measured. In MΦ stimulated with LPS, the mRNA expression levels of IL-6 and TNF-α were upregulated A. MΦ werUC is an intractable disease specified by the Ministry of Health in Japan, and the mechanism underlying its onset remains unclear. In this context, the lack of therapeutic targets may be the strongest and most significant hurdle to the establishment of a treatment technique for this disease in the field of clinical medicine. In addition, the continuous increase in the number of UC patients is currently a concern worldwide.UC reportedly occurs in the rectal region of the large colon; however, its origin remains unclear. The purpose of the present study was to identify novel biomarkers and therapeutic targets for UC using an experimental animal model. First, changes in the levels of some proteins in the rectal tissue of UCR were investigated, and the expression of some components was significantly different between WT and UCR rats. Among them, the most noticeable protein was CA-III, which encouraged us to proceed with scientific research to determine the origin of UC. The intracellular concentration of CA-III was significantly decreased in MΦ, although the reason for this is unclear. We previously reported the possibility that S100 proteins are deeply involved in the onset and aggravation of UC and that immunological assessments of the balance between S100A8 and S100A9 may be used to establish the current state of MΦ in the gastrointestinal tract . FurtherNevertheless, the column enabled us to compare, to some extent, the results obtained by SDS-PAGE using the rectal proteins and to select protein bands with different expression levels between WT and UCR. The levels of eight protein bands (B1–B8) appeared to decrease in the rectal region of UCR, while the levels of three protein bands (B9–B11) increased . Six proCarbonic anhydrases (CAs) are zinc-containing enzymes that reversibly catalyze the hydration of carbon dioxide in the body to maintain homeostasis . Previou2-macrogulobulin concentrations and DAI scores was also evaluated, with R-values of 0.581 and 0.474, respectively [Immunohistochemical staining showed that the mucosal epithelium in the rectal region of WT abundantly expressed CA-III, whereas its amount decreased with the aggravation of inflammation in the rectum of UCR A. Many iectively . The R-vIn that in vitro study, CA-III was abundantly expressed in normal MΦ but to a lesser extent in MΦ stimulated with LPS . In UC, The measurement of serum CA-III levels is useful for diagnosing UC in early stages. In the future, we intend to measure serum CA-III concentrations in patients with mild-to-severe UC. The clinical application of CA-III makes it challenging to clarify the possible use of CA-III as a therapeutic agent for UC. To confirm its pharmacological effects in fundamental research, a large amount of purified CA-III may be needed to treat the inflamed rectal tissues of UCR. We found that CA-III reflects the severity of experimental colitis. Thus, serum CA-III level may be a useful biomarker for experimental UC. In addition, CA-III in the colonic mucosa and MΦ significantly suppressed the secretion of inflammatory cytokines and protected against aggravation of experimental UC. Therefore, CA-III may be an important tool for investigating the causes of UC.


Head and neck squamous cell carcinoma (HNSCC) is one of the worst and most common malignant tumors. This study is aimed at studying the complex interaction between glycosylation-related genes and HNSCC.  The Cancer Genome Atlas (TCGA) contains gene expression profile data of HNSCC and normal tissues, as well as patient survival and clinical data. Combining five glycosylation-related gene sets, bioinformatics was used to analyze the expression of glycosylation-related genes in TCGA-HNSCC datasets and to identify prognostic risk markers, analyze their prognostic value, and the influence of glycosylation-related genes on the tumor immune microenvironment. P < 0.001). ROC analysis was used to evaluate the prognostic efficacy of prognostic risk markers, and the results showed that the prognostic risk markers had good efficacy in predicting the prognosis of patients. We also found that there is a correlation between glycosylation-related genes, PD-L1, and immunocyte infiltration, and there is a dynamic effect between the change in the copy number of glycosylation-related genes and the number of tumor-infiltrating immune cells.  Gene expression profiles and corresponding clinical information of 499 cases of HNSCC and 44 cases of adjacent tissues were obtained. Using 11 glycosylation-related genes to construct a prognostic risk score, the Kaplan-Meier curve analysis found that the overall survival of the high-risk group was significantly different than that of the low-risk group ( Our research shows that glycosylation-related prognostic risk markers may be independent risk factors for the prognosis of HNSCC. We have found that there may be subtle links between glycosylation-related genes, PD-L1, and immunocyte infiltration, which has certain significance for exploring the occurrence and development of HNSCC and exploring the research of targeted therapy. Head and neck cancer is one of the main causes of global morbidity and mortality; among which more than 90% are squamous cell carcinomas, and most are derived from the stratified squamous mucosa of the lips, oral cavity, oropharynx, hypopharynx, and epithelial larynx . CurrentThe research found that mechanisms related to abnormal glycosylation play an important role in promoting the occurrence and development of HNSCC . The stuAbnormal glycosylation is one of the unique characteristics of cancer cells. Specific glycan changes and abnormal glycosylation processes are crucial in tumorigenesis and metastasis . Studiesn = 499) and normal tissues (n = 44) and the corresponding clinical-related information were obtained.The genome-wide expression profiles and clinical data of patients with HNSCC were extracted from The Cancer Genome Atlas (TCGA) dataset. Gene profile expression comparing HNSCC tissues  package was necessary for the statistical analysis of this study. Each patient was assigned a unique risk score based on the linear combination of gene expression levels: Risk score = expression value of gene 1 × P value less than 0.05 was considered statistically significant.Using the risk score value to obtain the median as a cut-off value, 499 patients with HNSCC were divided into high- and low-risk groups. The Kaplan-Meier curve was used for survival analysis, and ROC analysis was used to predict the prognostic efficacy of the labels. Univariate and multivariate Cox regression analysis was used to analyze the correlation between predictive signatures of glycosylation-related genes and other clinical variables. A cBioPortalc is an online analysis database that provides visualization tools for research and analysis of cancer genetic data and uses molecular data obtained from cancer tissue and cytology to understand heredity, epigenetics, gene expression, and proteomics. STRING version 11.0 is an online tool that can modify and integrate information from many sources to analyze interactions between glycosylation-related genes.https://portal.gdc.cancer.gov/repository). We obtained and screened glycosylation-related gene sets on the GSEA website (https://www.gsea-msigdb.org/gsea/login.jsp), and we used five different gene sets  to explore whether these five glycosylation-related gene sets are different between the paracancer sample and the tumor sample. We found that the abovementioned gene set in HNSCC was significantly different than that of the adjacent tissues and tumor tissues  of each prognostic risk index was calculated by multivariate Cox analysis. The unique risk score of these patients is obtained based on the regression coefficient and the expression level, where the risk score value (Risk score) = (PSMC1 expression × 0.549384) + (NAGK expression×−0.35266) + (AREG expression × 0.133426) + (DDOST expression×−0.31938) + (ATP6V1E1 expression × 0.424889) + (PLOD2 expression × 0.149545) + (TMED10 expression×−0.34939) + (ALG5 expression × 0.323277) + (ARF3 expression × 0.617688) + (OST4 expression × 0.407259) + (KDELR1 expression × 0.37953). Based on unique risk score, HNSCC patients were divided into a high-risk group and a low-risk group according to the median value of the risk score, with a risk score greater than the median being classified as a high-risk group (n = 249) and a risk score less than the median being classified as low-risk risk group (n = 250). The Kaplan-Meier (KM) survival curve was used to assess the prognostic difference between the two groups. The results showed that the OS of the high-risk group was significantly different from that of the low-risk group  (The regression coefficient (< 0.001) . To test< 0.001) , indicat< 0.001)  and founIn the cBioPortal online database, we analyzed the mutations of 11 glycosylation-related genes in HNSCC through clinical samples. The results showed that 78 patients (15.6%) had genetic mutations. Among them, the rate of mutation of the PLOD2 gene was 9%, including 37 cases of amplification and 8 cases of missense mutations. PSMC1 and ATP6V1E1 gene mutations accounted for 1.4%, TMED10 gene mutations accounted for 1.2%, and ALG5 gene mutations accounted for 1%; in addition, NAGK, AREG, DDOST, KDELR1, ARF3, and OST4 gene mutation rates were all lower than 1% .P < 0.001 for PSMC1, NAGK, DDOST, ATP6V1E1, KDELR1, PLOD2, TMED10, and ARF3; P = 0.013 for AREG; P = 0.002 for ALG5; P = 0.03 for OST4)   . We clasctively) . For PSMctively) , but theThe Pearson correlation coefficient was used to test the strength of the coexpression of glycosylation-related genes. As shown in P < 0.001, 95% CI 1.221-1.465, HR = 1.337). The results of univariate and multivariate Cox regression analysis showed that prognostic risk signature composed of 11 glycosylation-related genes can predict the prognosis of patients and can be independent of clinical characteristics. In summary, prognostic risk markers can be applied to predict OS in patients with HNSCC  and low-risk subgroups (n = 82); while patients younger than 65 years old have high risk subgroup (n = 156) and low-risk subgroup (n = 168), there was a significant difference in OS between the two subgroups (P < 0.001). Patients were stratified by sex; for the females, 64 were in the high-risk subgroup and 69 were in the low-risk subgroup, while for males, there were 185 cases in the high-risk subgroup and 181 cases in the low-risk subgroup. The analysis showed that there were significant differences in OS rates between the two subgroups (P < 0.001).We carried out a stratified analysis of each clinical feature and further evaluated the correlation between the risk score and the clinical feature. The result is shown in n = 359) and GIII/GIV (n = 121). According to the risk score, there were 180 cases in the high-risk subgroup and 179 cases in the low-risk subgroup for GI/GII. We found a significant difference in OS between the two subgroups (P < 0.001). The same results were also observed in the two subgroups of patients with GIII/GIV tumors (P = 0.003). Patients were divided into T1 and T2 groups (n = 177) and T3 and T4 groups (n = 266) based on the T stage of the tumor. The survival rate of the low-risk subgroup in the T1 and T2 groups was significantly different from that of the high-risk subgroup (P = 0.003), and similar survival rates of the high- and low-risk subgroups in the T3 and T4 groups were also found (P < 0.001). Depending on the presence or absence of lymph node metastases, these patients were divided into two groups. We found that the survival rates of the low-risk and high-risk subgroups were significantly different between the two groups . We divided patients into stage I and II groups (n = 94) based on the clinical stage of the tumor, which was high-risk subgroups (n = 36) and low-risk subgroups (n = 58). Stages III and IV were grouped together (n = 337), for the high-risk subgroup (n = 184) and low-risk subgroup (n = 153). In tumor stage III and IV groups, the survival rate of patients in the high-risk subgroup was significantly different from that of patients in the low-risk subgroup (P < 0.001). In the tumor clinical stages I and II groups, there was no significant difference in the survival rate of patients in the two subgroups (P = 0.125). Since there was only 1 patient with distant metastasis, we could only stratify patients without distant metastasis to obtain a high-risk subgroup and a low-risk subgroup. The results suggested that there was a significant difference in survival rate between the two groups (P < 0.001).All patients were stratified according to tumor grade (G) and were divided into two subgroups: GI/GII  based on glycosylation-related genes on immune cell infiltration were further analyzed. The CNA of identified glycosylation-related genes significantly influenced the infiltration of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells in HNSCC. These results indicated that glycosylation-related genes had a key regulatory effect on the TIME in HNSCC patients .The occurrence, development, and invasion of tumors are accompanied by changes in glycosylation of related glycoproteins. The changes in the structure of carbohydrates on the surface of cancer cells play an important role in the course of cancer . The proAbnormal glycan structure and glycosylation process may be an important factor in the complex process of immune escape of tumor cells . The proImmune-related cells are considered an indispensable key part of the innate and adaptive immune system, such as B cells, T cells, dendritic cells (DCs), and natural killer (NK) cells. During tumorigenesis, abnormal glycosylation processes can directly or indirectly affect the immune response of tumor cells, thereby regulating tumor progression . ExploriThe small sample size in the validation data set underlines the limitations of this study and suggests there may be selection bias in the results. To verify the reliability of the conclusions, more prospective clinical trials are necessary. In addition, our study only explored the potential correlation of glycosylation-related genes, PD-L1 and immune infiltration in the occurrence and development of HNSCC, more detailed studies are needed to analyze the specific mechanisms of glycosylation-related genes in promoting HNSCC progression. It is necessary to further study how glycosylation-related genes, PD-L1 and immune infiltration, jointly regulate the occurrence and development of tumors. It provides clinicians with more reliable diagnostic markers, provides an important basis for finding potential targets for HNSCC treatment, and is expected to provide precise and personalized treatment strategies for HNSCC patients.Our study identified an 11 glycosylation-related gene signature of prognostic risk markers, namely, PSMC1, NAGK, AREG, DDOST, ATP6V1E1, KDELR1, PLOD2, TMED10, ALG5, ARF3, and OST4. These markers can predict the prognosis of patients with HNSCC, indicating that glycosylation-related genes can be used as prognostic risk markers for HNSCC. We analyzed and evaluated the correlation between 11 glycosylation-related genes and PD-L1, as well as the potential regulatory mechanisms. The risk score of glycosylation-related genes is significantly related to the level of immune cell infiltration in HNSCC patients, and glycosylation-related genes may be involved in the regulation of the immune microenvironment of HNSCC. Therefore, identifying glycosylation-related genes and molecular pathways that affect tumor immune response and further study into their regulatory mechanisms may provide potential molecular targets for improving the sensitivity of HNSCC to targeted immunotherapy.


The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has been leading to dramatic health, social and economic problems around the world. It was necessary to introduce worldwide vaccination program against SARS-CoV-2 virus. Vaccination of billions of people around the world leads to many questions about risk of vaccines and possible side effects. It is well known that acute disseminated encephalomyelitis (ADEM) is a rare, but possible complication of vaccines. Previously, cases of ADEM following various COVID-19 vaccines, including the vaccines from AstraZenica, Pfizer, Sputnik V, SinoVac, Moderna, Sinopharm, have been described. In this case report, we present the first documented case of ADEM following the COVID-19 vaccine Ad26.COV2.S from Johnson & Johnson.We present the case of a 31-year-old female with gradually progression of right-sided weakness and numbness during a three-week period. Four weeks prior to symptom onset, the patient received the single-dose SARS-CoV-2 vaccine Ad26.COV2.S. Neuroimaging revealed five large juxtacortical T2 FLAIR hyperintense lesions with incomplete contrast enhancement on post-contrast T1 images located supratentorial: one in the right cerebral hemisphere and four in left cerebral hemisphere. The patient was followed up for four months. Symptom debut, clinical picture and MRI were typical for ADEM and the patient completely recovered after high dose intravenous methylprednisolone treatment.This is, to the best of our knowledge, the first case report of ADEM following the COVID-19 vaccine Ad26.COV2.S. This case illustrates, although ADEM is a rare complication following SARS-CoV-2 vaccines, the necessity of maintaining a vaccine safety monitoring system to identify patients at high risk from developing severe complications from the vaccines. A variety of vaccines have been granted emergency use authorization to fight the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic, also referred to as the COVID-19 pandemic. In the post-authorization phase, a broad range of neurological complications have been reported including neurovascular, neurometabolic and neuroinflammatory diseases , , due to previous HBV vaccination.6/L (reference range 0–5 × 106/L); protein levels were 0.31 g/L (reference range 0.15–0.5 g/L); IgG index was 0.47 (reference range 0.38–0.67); and oligoclonal bands in CSF and blood were negative.Pharyngeal swab was negative for SARS-CoV-2 on reverse-transcriptase polymerase chain reaction assay. The cerebrospinal fluid (CSF) white blood cell count was 1 × 10Brain and spinal cord magnetic resonance imaging (MRI) revealed five large juxtacortical T2 FLAIR hyperintense lesions with incomplete contrast enhancement on post-contrast T1 images located supratentorial: one in the right cerebral hemisphere and four in left cerebral hemisphere. The largest lesion was in the right frontal lope with a diameter of approximately 4.4 cm  or the European Medicines Agency (EMA) as it has been assessed that the overall benefits of the vaccines outweigh their risks. However, due to the extensive global use of SARS-Cov-2 vaccines. it is important to ensure a fast-acting safety monitor system to discover the rare complications to the vaccines, e.g., ADEM. This would improve the likelihood of identifying the right patients who are at high risk from developing severe complications from the vaccines.The manifestation of the clinical and paraclinical findings in this case did meet the criteria for ADEM (Krupp et al. During the four-month follow-up, the clinical and paraclinical features followed a monophasic disease course; however, a prolonged observation is required to confirm this.Overall, we have identified 17 previous case reports of patients with ADEM following SARS-CoV-2 vaccination, including vaccines based on mRNA, viral vector and inactivated virus (Kania et al. In previous cases, the patients age ranged from 19 to 88 years but were predominantly in the ages between 30 and 50, and the majority was females, which is in line with our case. In contrast, the previous cases primarily had time from vaccination to symptoms of 10–14 days, which is half the time as the patient in our case. Additionally, most patients from the previous cases, similar to the patient in our case, had no previous significant medical history, and none of the patients were treated with immune-modifying drugs. This illustrates, although ADEM is a rare complication following SARS-CoV-2 vaccines, the necessity of maintaining a vaccine safety monitoring system to identify patients at high risk from developing severe complications from the vaccines.


Trends from around the world suggest that the omicron BA.2 subvariant is increasing in proportion to the original BA.1 subvariant. Here we report two cases of co-infection with omicron BA.1 and omicron BA.2 in co-exposed individuals. In both individuals, genome sequencing and/or S-gene specific PCR identified omicron BA.1 at early time-points, which was replaced by omicron BA.2 at later time-points of the infection. The timeline of our data supports the proposition that BA.2 outcompetes BA.1 in a real-life scenario, and in time becomes the dominant variant in the upper respiratory tract of the host. Throughriants/) . The VOCriants/) . Severalriants/) . In lineTwo individuals with COVID-19 symptoms were confirmed to be positive by PCR and then analyzed by genome sequencing at different time points at our institution . First, Her partner, a 35-year-old male  tested positive at day 1 for trace amounts of SARS-CoV-2, yielding a non-SGTF PCR, which was confirmed by follow-up non-SGTF PCRs at day 2, day 4, day 8, day 11, and Day 14 . He expeBoth individuals were fully vaccinated with two doses of the BNT162b2, COVID-19 mRNA vaccine, 9 and 11 months prior to the infection, respectively. The most likely route of omicron BA.1 infection for individual 1 was a beauty salon; individual 2 was most likely exposed to omicron BA.2 in a crowded restaurant. Both individuals underwent home isolation together and experienced mild symptoms. Blood work results at day 4 were within reference ranges.For the detection of viral RNA by RT-PCR and sequencing, nasal and oropharyngeal swabs were collected for each time point. Both swabs were combined and immersed in saline solution and processed immediately. RNA was extracted with the abGenix  automatic DNA/RNA extractor, and PCR was carried out on QuantStudio™ 5  thermal cycler using the TaqPath protocol , according to the manufacturer’s recommendations. Positive and negative controls were routinely included in each run.PCR tiling of COVID-19 virus” (Version: PTC_9096_v109_revD_06February 2020). The protocol was modified with rapid barcoding instead of native barcoding using the Rapid Barcoding Sequencing Kit, SQK-RBK004 . The samples were sequenced on Flow Cell R9.4.1 using the MinION device.Reverse transcription using SuperScript IV Reverse Transcriptase  and SARS-CoV-2 genome amplification using the ARTIC panel of primers  was performed as described in the Nanopore protocol “medaka consensus pipeline for creating a consensus sequence using Galaxy platform (https://clades.nextstrain.org/). Genome coverage was between 23x and 121x . Multiple FASTQ files were concatenated into one, and genome assembly was conducted with the platform . Consensand 121x  with twoand 121x  was evaland 121x .In this report, we illustrate two interesting cases of co-infection with omicron BA.1 and omicron BA.2 in co-exposed individuals in the same household. In both individuals, genome sequencing and/or S-gene specific PCR identified omicron BA.1 at early time-points, which was replaced by omicron BA.2 at later time-points of the infection.The timeline of our sample collection and symptom onset supports the proposition that individual 1 initially got infected with omicron BA.1 and exposed individual 2. During the incubation phase or early phase of infection individual 2 most likely got exposed to omicron BA.2 independently, which quickly outcompeted omicron BA.1 in his upper respiratory tract. During home isolation with individual 2, individual 1 who was already infected with omicron BA.1 got exposed to omicron BA.2, which seems to have outcompeted omicron BA.1 in the following days. These observations suggest that omicron BA.2 has biological properties allowing it to outcompete omicron BA.1 in the host, at least in the immunological and genetic context of these two individuals. Similar cases of co-infection with two different SARS-CoV-2 variants could serve as an evolutionary substrate for viral recombination events, and the emergence of new variants.In addition, our study has several limitations. The most obvious one is the small sample size of two non-related individuals; meaning that our observations are not readily translatable for other households or larger populations. Second, we were unable to provide complete ungapped genome assembly and full genome coverage of the sample collected on day 2 from individual 2. Unfortunately, this prevented us to conduct more detailed genomic analyses to evaluate the complete distribution and representation of the omicron subvariants in that sample.All in all, our observations although limited in nature are consistent with the epidemiological situation in several other countries, where omicron BA.2 has replaced or has been replacing omicron BA.1. These studies along with our observations suggest that omicron BA.2 has biological features leading to host-specific growth advantage.


We hypothesised that the clinical characteristics of hospitalised children and young people (CYP) with SARS-CoV-2 in the UK second wave (W2) would differ from the first wave (W1) due to the alpha variant (B.1.1.7), school reopening and relaxation of shielding.Prospective multicentre observational cohort study of patients <19 years hospitalised in the UK with SARS-CoV-2 between 17/01/20 and 31/01/21. Clinical characteristics were compared between W1 and W2 .2044 CYP < 19 years from 187 hospitals. 427/2044 (20.6%) with asymptomatic/incidental SARS-CoV-2 were excluded from main analysis. 16.0% (248/1548) of symptomatic CYP were admitted to critical care and 0.8% (12/1504) died. 5.6% (91/1617) of symptomatic CYP had Multisystem Inflammatory Syndrome in Children (MIS-C). After excluding CYP with MIS-C, patients in W2 had lower Paediatric Early Warning Scores , lower antibiotic use and less respiratory and cardiovascular support than W1. The proportion of CYP admitted to critical care was unchanged. 58.0% (938/1617) of symptomatic CYP had no reported comorbidity. Patients without co-morbidities were younger , had lower PEWS, shorter length of stay and less respiratory support.We found no evidence of increased disease severity in W2 vs W1. A large proportion of hospitalised CYP had no comorbidity.No evidence of increased severity of COVID-19 admissions amongst children and young people (CYP) in the second vs first wave in the UK, despite changes in variant, relaxation of shielding and return to face-to-face schooling.CYP with no comorbidities made up a significant proportion of those admitted. However, they had shorter length of stays and lower treatment requirements than CYP with comorbidities once those with MIS-C were excluded.At least 20% of CYP admitted in this cohort had asymptomatic/incidental SARS-CoV-2 infection.This paper was presented to SAGE to inform CYP vaccination policy in the UK. A small proportion of CYP exposed to the virus go on to develop a severe hyperinflammatory syndrome2 known as multisystem inflammatory syndrome in children (MIS-C), also known as paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS), which shares features of Kawasaki disease and Toxic Shock Syndrome, and often requires management in intensive care.7Children and young people (CYP) were significantly less affected than adults during the first wave of the COVID-19 pandemic, with regards to case numbers, disease severity, hospital admissions and death.8 A significant amount of knowledge was gained about the clinical characteristics and outcomes of COVID-19 in CYP during the first wave of the pandemic, however, several external factors changed with the emergence of the second wave. Most UK schools were closed during the first pandemic wave in the spring and summer of 2020, with a few remaining open for children of key workers but were mostly open during the subsequent autumn and winter wave of infection  and the second wave from the beginning of September 2020 to the end of April 2021 (peaking in early January).nfection . This po14 This may have been due to the emergence of the variant coinciding with a period when schools were open and subject to increased testing, but the rest of UK society was in “lockdown”.12 Whether the alpha variant, dominant in the second wave, causes different symptoms or more severe disease in CYP compared to strains circulating in the first wave has not been analysed in detail.The emergence of the alpha variant in England also led to concerns of increased transmissibility in CYP as they formed a higher proportion of total cases in England when compared to the first pandemic wave.We test the hypothesis that clinical characteristics of hospitalised children with SARS-CoV-2 in the UK second wave would differ from the first due to the combined impact of the alpha variant, school reopening and relaxation of shielding.We aimed to characterise and compare the clinical features and outcomes of CYP aged <19 years who were hospitalised with SARS-CoV-2 infection during the first and second waves across England, Scotland, and Wales from 17th January 2020 to 31st January 2021, as part of the International Severe Acute Respiratory and Emerging Infection Consortium -World Health Organisation, Clinical Characterisation Protocol in the United Kingdom (ISARIC WHO CCP-UK).https://isaric4c.net.15 We included all patients aged <19 years with clinician-reported SARS-CoV-2 infection who were enroled into the ongoing, prospective ISARIC WHO CCP-UK cohort study involving National Health Service (NHS) hospitals in England, Wales, and Scotland between 17th January 2020 to 31st January 2021 who had at least 2 weeks of outcome data available .16 Patients were managed by their local clinicians and participation in this study had no influence on management. We used the Strengthening the Reporting of Observational Studies in Epidemiology guidelines for reporting this observational study.The protocol, associated documents, and details of the Independent Data and Material Access Committee (IDAMAC) are available at Data were collected from healthcare records onto case report forms through a secure online database, REDCap . Demographic  and baseline data (including comorbidities and regular medications taken) alongside data on symptoms, clinical signs, laboratory and pathology investigations, and treatments received while admitted were collected. Centres also recorded whether their team had treated patients as having MIS-C.Patients were included in this report if the study team had reported them as ‘laboratory-proven’ SARS-CoV-2. Where patients were reported as ‘suspected’ SARS-CoV-2, the patients’ virological data were reviewed, and patients were included if there was documented evidence of a positive PCR, serology or lateral-flow antigen testing for SARS-CoV-2 .Supplementary Methods). Patients in whom SARS-CoV-2 was judged to be incidental or who were asymptomatic at the time of assessment for SARS-CoV-2 were censored from the main analysis.We reviewed all available free text for evidence of incidental SARS-CoV-2 infection  was used as a measure of disease severity at admission.The primary outcomes of this study were admission to critical care (high dependency units (HDUs) or paediatric intensive care units (PICUs)), development of MIS-C, and in-hospital mortality for CYP and young people with symptomatic SARS-CoV-2 infection (COVID-19). We also examined the need for any respiratory and cardiovascular (inotropic) support.Supplementary Results).As specialist children’s hospitals (tertiary centres) are more likely to have both paediatric critical care facilities and paediatric research teams with capacity to support participating in the study, it is possible that CYP admitted to these centres are over-represented. We compared the proportion of CYP who were reported from hospitals with onsite access to a PICU to ascertain whether this differed between the waves, potentially influencing the severity of patients reported (see Supplementary Results).To explore how well the ISARIC data reflected regional variations in SARS-CoV-2 prevalence, we also compared the number of CYP reported to ISARIC against the numbers of local SARS-CoV-2 cases identified by pillar 1  and pillar 2 (community) testing across NHS regions . As such, given the time lag in the presentation of MIS-C, we anticipated the number of cases of MIS-C in the second wave would be an underestimate. This could bias towards fewer severely ill CYP in the second wave. To reduce this bias, patients with MIS-C were censored for analyses comparing disease severity or treatments received across waves 1 and 2, but retained for whole cohort analyses.Our previous analysis identified a peak of MIS-C cases occurring approximately four weeks after the peak of CYP admissions in the first wave, with patients with MIS-C being five times more likely to be admitted to critical care.t, analysis of variance, Mann-Whitney U, or Kruskal-Wallis tests, according to data distribution. We compared categorical data by using χ2 tests and considered a p value below 0.05 to be statistically significant. All tests were two sided and we made no adjustment for multiple comparisons. A directed acyclic graph was constructed prior to undertaking a mixed effect multivariable analysis (Continuous variables are displayed as means (standard deviations) or if non-normally distributed as medians (interquartile ranges). Categorical variables are presented as frequencies (percentages) unless otherwise stated. For univariable comparisons, we used Welch’s analysis . HospitaPatients and the public were not involved in the design, conduct, or reporting of this rapid response research which is part of an ongoing urgent public health research study, however their involvement is in now progress.In England and Wales routine anonymised data from medical records was collected without the need for consent under regulation 3 (4) of the Health Service (Control of Patient Information) Regulations 2002. In Scotland, a waiver of need for consent was obtained from the Public Benefit and Privacy Panel. Ethical approval was given by the South Central–Oxford C Research Ethics Committee in England (reference 13/SC/0149) and the Scotland A Research Ethics Committee (reference 20/SS/0028).Between 17th January 2020 and 31st January 2021, 187,267 admissions of all ages were enrolled. There were 2044 (1.1%) CYP aged <19 years of age with clinician-reported SARS-CoV-2 infection reported from 187 hospitals across England, Scotland, and Wales. Of these, 1540 (75.3%) had symptoms at presentation, 427 (20.6%) had asymptomatic or incidental SARS-CoV-2 infection and 77 (3.8%) were missing data on symptoms  (Table 1). CYP of South Asian ethnicity were over-represented in W2  compared to W1 . W2 saw a lower proportion of likely hospital-acquired SARS-CoV-2 (2.2% (21/952) vs W1, 6.9% (46/665), p < 0.001). Fever was more common in W1 (76.8% (491/639) vs 63.6% (544/855), p < 0.001, In total, 764 CYP were admitted during wave one  and 1280 during wave two . CYP in W2 were significantly older  than W1 (4.0 (0.4–13.6), p = 0.005, Table p = 0.989, Table p < 0.001)), were less likely to receive high flow oxygen, non-invasive or invasive respiratory support as well as fewer patients in W2 requiring inotropic support (1.0% (8/780) vs 3.6% ). CYP in W2 were more likely to receive oral steroids. These associations persisted in a sensitivity analysis of the whole cohort (Paediatric Early Warning Score (PEWS) at presentation was lower in W2 than W1, with 41.2% (343/832) of CYP in W2 having a PEWS > 2 at presentation vs 48.9% in W1 . CYP with MIS-C in W2 were less likely to receive IVIg than in W1 (59.5% (25/42) vs 83.7% (41/49), p = 0.018, There were 163 potential MIS-C patients, of whom 91 were confirmed by sites, 46 had other diagnoses and there was no response for 26 patients . There wWe reviewed the demographics and key clinical characteristics of CYP admitted to critical care, excluding those with asymptomatic or incidental SARS-CoV-2 infection (but including those with MIS-C). On univariable analysis, age groups 5-9 and 10-14 years were associated with critical care admission, as was non-white ethnicity, hospital-acquired SARS-CoV-2 infection, PEWS > 2 at admission and presence of an underlying comorbidity . Of the Methods), these patients were excluded from the multivariable analysis to reduce bias when comparing severity between the waves. Neonates and CYP aged 10-14 years and 15-19 years were more likely to be admitted to critical care  and an overall mortality rate of 0.8% (12/1504). Information was available for 11 of these deaths. All 11 had significant comorbidities . Five were <5 years old and six were >15 years old.p = 0.014), and a shorter length of stay (2.0 days (1.0–3.0) vs 3.0 days (1.0–7.0), p < 0.001). CYP with no reported comorbidities were less likely to receive antiviral therapy, steroids, and all forms of respiratory support than in CYP with comorbidities (Table Patients without a reported comorbidity made up 58.0% 938/1617) of the symptomatic cohort . Of CYP 8/1617 ofes Table .Table 3Tp < 0.001) in the second wave , have a reported comorbidity (51.3% (219/427) vs 43.7% (673/1540), p = 0.006) and evidence of an alternative reason for admission  vs 5.3% . They also had a lower median PEWS on presentation (1.0 (IQR 0.0–2.0) vs 2.0 (IQR 1.0–4.0), p < 0.001). No differences were seen in sex or ethnicity or IMD.We observed a significant increase in the proportion of patients who were asymptomatic at the time of SARS-CoV-2 detection from 10.4% (78/751) in the first wave to 24.7%  had asymptomatic or incidental SARS-CoV-2 infections. Of the symptomatic CYP, 5.6% (91/1617) had MIS-C and 16.0% (248/1548) were admitted to critical care. Within the symptomatic group, 0.8% (12/1504) died. There were 665 symptomatic CYP in W1 and 952 in W2, with those in W2 being older, more likely to be of South Asian ethnicity and with a lower proportion of hospital acquired SARS-CoV-2. Reassuringly, there was no evidence of a negative impact of relaxation in guidance to ‘shield’ vulnerable CYP in the latter part of 2020,19 Whilst there was no difference in PEWS at presentation, respiratory or cardiovascular support in CYP with MIS-C between the waves, those in W2 were less likely to receive IVIg. This may reflect increases in clinician confidence, local guidance, or decreased availability of the therapy.Despite concerns about more severe disease and fatalities associated with the alpha variant in adults, relaxation of shielding advice and increases in face-to-face schooling, no difference was found in the proportion of symptomatic CYP admitted to critical care between the two waves, instead pointing to less severe disease in W2. After excluding patients with MIS-C, CYP admitted during W2 had a lower PEWS at presentation, lower antibiotic use and less respiratory and cardiovascular support compared to W1. Oral steroid use was higher in W2, likely because of changes in national guidance adopting the results of the RECOVERY trial.2 with neonates and ages 10–14 and 15–19 years associated with admission in addition to ethnicity, PEWS at presentation and number of comorbidities.After exclusion of CYP with MIS-C, factors associated with admission to critical care remained very similar to our first report,Of particular interest were CYP without comorbidities. Those under 1 year comprised 42.4% of CYP admitted without comorbidities - an age group commonly admitted for brief periods of observation for viral illnesses. CYP with no reported comorbidities had a lower PEWS at presentation, shorter length of stay and received less respiratory support. However, 12.9% (112/876) of all hospitalised CYP without comorbidities were admitted to critical care, with 53 of these having MIS-C. CYP with MIS-C were also responsible for much of the invasive and non-invasive ventilation, inotrope use and IV steroids in the CYP group without comorbidities. Overall, the majority of children in hospital with symptomatic infection had no reported comorbidities (58.0% (938/1617). Of these CYP without reported comorbidities, 70.4% (660/938) were ≤ 11 years, representing a significant group in whom there is no current licenced vaccine available, while 47.5% (278/585) of CYP of vaccine-licensed age had no reported comorbidities. This suggests that targeting prevention strategies, such as vaccines, on the basis of comorbid status or risk groups will have somewhat limited impact on hospitalisation in CYP of vaccine licensed age and overall.The ISARIC WHO CCP-UK study provides an extensive, detailed and prospective dataset, continuously collected since the start of the pandemic and is uniquely placed to monitor changes in the characteristics of hospitalised CYP with SARS-CoV-2 in the UK from emergence to evolution of the pandemic. Unlike routine and population-based studies, data collection for ISARIC is focused on characterisation of novel emerging disease. Due to the need to retain a ‘lite’ data collection process, some data terms (particularly comorbidities) are only relevant to adults and not relevant to CYP. Reporting to ISARIC is voluntary and is likely to lead an underestimate in the number of asymptomatic or incidental cases reported, as some sites may have chosen only to report ‘true’ COVID-19 cases. Genotype data was not available.It is important to recall that the study definition of critical care is not just intensive care but includes high dependency units in secondary care centres. In paediatrics, patients are often admitted to these wards for close observation, without intensive therapy. This is borne out by our study finding much lower rates of high-level respiratory or cardiovascular support than rates of critical care admission.Curtailment of analysis on 31st January 2021, after the peak of the second wave and with community cases falling, was planned due to the urgent need to provide paediatric data on the alpha variant to inform public health policy at that time. However, MIS-C typically presents 2-4 weeks after infection, and therefore cases of MIS-C due to infection acquired in the second wave will be underestimated.A major strength of our study is that it highlights the importance of differentiating whether CYP are hospitalised because of COVID-19 i.e., disease caused by SARS-CoV-2 infection or with SARS-CoV-2 infection that was incidental. Analyses performed without excluding CYP where SARS-CoV-2 is incidental will produce misleading results. Describing and identifying factors predicting severity of COVID-19 could be biased in either direction by inclusion of both a high proportion of asymptomatic, or incidental infections, and inclusion of CYP admitted to critical care for unrelated reasons such as trauma.20 and death,22 although estimates of case-fatality rates may be limited by confounding factors.21 However, most studies did not include CYP, and reported risks appear to be age dependent. In a large community-based UK retrospective cohort, Nyberg et al. found an increased risk of hospital admission and mortality in adults older than 30 years testing positive for the alpha variant, but found no difference for young people under 20 years.20 In a brief report, Brookman et al. compared characteristics of 20 CYP admitted to a single London hospital in W1 to 60 CYP in W2 with no difference in demographics or increase in severity of disease.23 Our larger and more detailed study supports these findings.Data collection before and after the emergence of the alpha variant provides reassuring evidence that clinical characteristics in CYP did not change over time coincident with the rise to dominance of this strain. There is evidence in adults that the alpha variant is not only associated with higher transmissibility, but also higher risk of hospital admission25 particularly in the discussion about vaccination of CYP. The granularity of our study allowed us to also examine CYP admitted to hospital without comorbidities in detail. While this group appears to be driven by infants with short hospital stays for brief observation, 12.9% of CYP admitted without comorbidities required critical care admission. CYP with MIS-C made up half of this group, but this also suggests that there may still be a group of previously well CYP with as yet unclear risk factors for critical care.Much of the focus of recent reports in SARS-CoV-2 in CYP aims to identify comorbidities associated with critical care admission,27 Brookman et al. reported a prevalence of asymptomatic/incidental infection in their cohort of 33%.23 Our finding that at least 21% of reported cases in our cohort were asymptomatic/incidental may be an underestimate given that reporting was voluntary and the variable use of free text to record these details.Understanding of asymptomatic SARS-CoV-2 infection has increased throughout the pandemic, particularly in CYP and it is clear that to be clinically useful, studies must identify CYP who have asymptomatic or incidental infection among those who are hospitalised. In our study, we found an increase in asymptomatic and incidental SARS-CoV-2 infections in W2 when compared to W1. Factors influencing this finding will include the introduction of routine testing of all hospital admissions and twice weekly lateral flow antigen testing in secondary schools (CYP ≥ 11 years). Two single-centre studies from the USA have reported that between 40 and 45% of their paediatric admissions with SARS-CoV-2 were either incidental or unlikely to be due to the virus itself.28 UK hospitals gradually moved from testing based on case definition  to universal testing of all admissions. Reduction in hospital-acquired infection may be due to improved infection control procedures, earlier detection of community acquired infection, or both.Differences in demographics and symptomatology between W1 and W2 may reflect changes in testing patterns, infection control and surveillance practices over the course of the pandemic.Accepting the limitations above, we provide evidence suggesting the emergence of the alpha variant did not lead to more severe disease in CYP in the UK. With the Delta variant now dominating in the UK, our study serves an exemplar of both the strengths and limitations of large hospital-based studies in informing immediate public health approaches to emerging new variants. The key strength of our study is in providing a granularity of individual patient data which allows us to look in detail at clinical presentations and outcomes, identify important sources of bias, and provide comprehensive data over time. The key limitation is that this nuanced approach takes time to perform and is outpaced by the rapid evolution of this pandemic. As a result, initial incomplete data is by necessity used to inform policy, while more accurate information may only be gained in retrospect.We urge other paediatric cohort studies to develop processes to define and record asymptomatic and incidental SARS-CoV-2 infection and differentiate this in analyses from COVID-19 disease. In addition, this study raises the possibility of as yet unidentified risk factors for critical care in CYP without comorbidities. As new variants of SARS-CoV-2 emerge, there is no guarantee that the generally mild disease observed in CYP to date will continue to predominate. Paediatricians and epidemiologists must remain vigilant in monitoring patterns of SARS-Cov-2 infection in CYP and develop more efficient systems to inform policy and clinical practice with speed and accuracy.Supplementary TableSupplementary Information


Hypertension is a serious threat to public health globally owing to its high prevalence and related complications. It is the main risk factor for cardiovascular disease, kidney disease, eye problems, and death. Self-care practices have been emphasized as a major element in reducing and preventing complications from hypertension. Thus, this study aimed to assess hypertension self-care practices and associated factors in Bale Zone, Southeast Ethiopia.A health facility-based cross-sectional study was conducted at three public hospitals from April 1 to May 31, 2021. Data were entered into Epi-Data version 4.6 and exported to Statistical Package for the Social Sciences (SPSS) version 25.0 for analysis. The study participants were characterized using descriptive statistics. The associations between self-care practice and independent variables were modeled using binary logistic regression analysis. Adjusted odds ratios with a 95% confidence interval were used to estimate the association between self-care practice and independent variables. The statistical significance of the association was declared at p < 0.05.This study involved 405 hypertensive patients, with a response rate of 96.7%. The overall level of good self-care practice was 33.1% . The multivariable logistic regression model showed that age under 65 years , good knowledge of hypertension self-care practice , absence of a depression  and good self-efficacy  were independent predictors of good self-care practice.The level of good hypertension self-care practice in the study area was low. Hence, it is crucial to expand non-communicable disease control programs and implement public health interventions on self-care for hypertension. Moreover, to enhance hypertension self-care practices, patient-centered interventions are essential. Hypertension is a significant public health challenge in both developed and developing countries because of its high prevalence and associated complications –3. SystoOne-third of adults worldwide have hypertension, and by 2025, that number is estimated to rise to 1.56 billion adults worldwide, with more than 125 million of those individuals living in sub-Saharan Africa –9. A sysHypertension is the main risk factor for cardiovascular disease, kidney disease, eye problems, and death , 11, 12.The actual cause of hypertension is uncertain, but research shows that risk can be increased by modifying factors like heavy alcohol use, lack of physical activity, high salt intake, smoking, and a higher body mass index, and avoiding these factors is an essential aspect of self-care practice , 9, 16. Patients must follow self-management protocols for hypertension to meet treatment objectives by raising the quality of life, preventing complications, and spending less on medical care –23. PatiAccording to previous studies various factors, including sex, age, educational status, place of residence, occupational status, socioeconomic status, time since diagnosis, comorbidity, source of information about self-care and knowledge of disease and treatment, self-efficacy, social support are all associated with components of hypertension self-care practice –29.Although there are few studies on self-care practices among hypertensive patients in Ethiopia, there is no evidence regarding hypertension self-care practices and associated in the study area. Due to this, it was determined that assessing self-care practices and associated factors might help in designing programs and developing strategies for efficient health education and patient empowerment as well as helping close knowledge gaps. Therefore, this study aimed to assess the level of self-care practices and associated factors among hypertensive patients in Bale Zone, Southeastern Ethiopia.To determine the level of hypertension self-care practices among adult hypertensive patients in public hospitals of Bale zone in 2021.To identify factors associated with hypertension self-care practices.An institutional-based cross-sectional study was conducted in the Bale Zone, Southeastern Ethiopia from April 1 to May 31, 2021. The Bale Zone is the second largest of the 20 zones in the Oromia Regional State, located 430 km from Addis Ababa, the capital city of Ethiopia. According to the Central Statistical Agency's 2007 population census, the zone's total population is estimated to be 1,402,492, with 688,975 females. The zone has five hospitals, 82 health centers, and 381 health posts. The current study was conducted in three public hospitals, namely Madda Walabu University Goba Referral Hospital (MWU GRH), Bale Robe General Hospital (BRGH), and Dello Menna General Hospital (DMGH).The study population consisted of all adult hypertensive patients on follow-up care at the selected public hospitals during the study period and those who met the inclusion criteria. The study included hypertensive patients aged 18 and older who had been on follow-up care for at least 6 months, based on diagnosis codes in the patient hospital records. Patients with cognitive impairment and those who were critically ill during the data collection period were excluded from the study.Zα/2 = 1.96, 95% confidence level, a margin of error (d = 0.045), and a proportion of good self-care practice (p = 0.299) from a previous study conducted in Harar [The sample size was calculated using a single population proportion formula assuming: in Harar . By consThe calculated sample size was proportionally allotted to the selected hospitals based on their preceding 3 months’ average client flow for hypertension follow-up during the study period. Accordingly, there were expected to be 855 hypertensive patients across the three hospitals during the study period  and the calculated sample size was distributed in accordance with this . Knowledge of hypertension and self-care. Psychosocial factors: social support, depression, and self-efficacy.Self-care practice was measured using the hypertensive self-care activity level effect (H-SCALE) . The scaThree items containing the number of days in the last 7 days assessed medication adherence. The responses were summed (range: 0–21), and the participants who reported that they had followed these three recommendations on all weekdays were considered adherents (score = 21), 38.Weight management was assessed using 10 items based on a 5-point Likert scale ranging from 1 (strongly disagree) to 5(strongly agree) with a sum ranging from 10 to 50. Patients who received 40 out of 50 total points were considered to be good weight management practice adherents .Two items were used to assess physical activity. The responses were then added up (range: 0–14). Participants were deemed physically active if they had a score of eight or higher .. Patients were classified as nonsmokers if they had not smoked or had not taken even one puff in the last 7 days [Smoking was assessed by one item whether the patients smoked or even a puff in the last 7 dayst 7 days .Twelve items were used to measure the low-salt diet. After the mean was calculated, a patient is considered adherent to the low-salt diet if they scored ≥ 6 .Three items were used to assess alcohol intake. When patients did not drink alcohol at all, they were regarded as abstainers , 39.The Hypertension Evaluation of Lifestyle and Management (HELM) scale was used to assess knowledge of hypertension. Patients were deemed to have adequate or good knowledge if their scores were at or above the mean .Self-efficacy was assessed using the Self-Efficacy to Manage Chronic Disease Scale (SEMCD). The SEMCD is a set of six measures ranging from 1  to 10 . If the patient responds to self-efficacy questions above the mean of six items, he/she was regarded as having good self-efficacy .Depression was screened using the patient health questionnaire-9 (PHQ-9). The PHQ-9 scale goes from 0  to 3 (nearly every day) The total number of points ranges from 0 to 27 and if the participant scores ≥ 10, he/she were considered to have depression .Social support was measured by the Oslo Social Support Scale (OSSS-3) containing three items: A four-point Likert scale with a range of 1 to 4 is used to rate the first item. Ratings for the second and third items are given on a five-point Likert scale with a range of 1 to 5. The total range of scores is between 3 and 14. Poor support was defined as 3 to 8, moderate support as 9 to 11, and strong support as 12 or higher .To assure the quality of the data, data collectors and supervisors were trained for 2 days on research ethics, data collection tools, and procedures. The questionnaire was first prepared in English and then translated into Afan Oromo and back to English to ensure consistency. We pretested the questionnaire on 5% of the total sample (21 hypertensive patients on follow-up) in Ginner Hospital to check for its validity likewise, we adjusted and rearranged some of the questions to ensure clarity, ease of understanding, and cohesion. Investigators and supervisors strictly supervised the data collection process.p-value of < 0.25 to be considered as potential candidate variables for multivariable logistic regression to control confounders. Hosmer and Lemeshow’s goodness-of-fit test was used to check the model adequacy, considering good fit at a p-value ≥ 0.05. Afterward, a multivariable logistic regression analysis was performed to identify factors associated with good self-care practices. Adjusted odds ratios (AOR) with a 95% confidence interval were used to report the results. Statistical significance was deemed at a p-value of < 0.05.The collected data were coded, cleaned, and reviewed for completeness to reduce data entry errors. The data were then entered into Epi-Data version 4.6 before being exported to the Statistical Package for the Social Sciences (SPSS) version 25.0 for analysis. The normality assumptions were checked to assess the distribution of continuous variables and choose the ideal summary measure. Descriptive statistics were done using frequency, percentage, mean, and standard deviation. Before regression analysis was conducted data were checked for multi-collinearity, normality, linearity, independence of residuals, and outliers, and no significant violations of the regression analysis's assumptions were identified. A bi-variable logistic regression model was used to identify those variables having a ) years. About 309(76.3%) of them were married, and 141 (34.8%) attended college and above  were males and 212 (52.3%) reside in an urban area. The mean age of the participants was 53  of the participants had medically confirmed other comorbid diseases, 92 (22.7%) had a family history of hypertension, and 193(47.7%) had a normal body mass index. In terms of the duration of hypertension diagnosis, 239 (59%) of participants were diagnosed less than 5 years ago and 209(51.6%) had a history of missed follow-up. About 254 (62.7%) of the participants had good knowledge about self-care practices for hypertension and 202(49.9%) of them got information about self-care practice from a healthcare professional. Among the participants, 168 (41.5%) had good self-efficacy, 102 (25.2%) had depression, and 148 (36.5%) had strong social support , determined from the six domains of self-care practice. About 198 (48.9%) of patients had good antihypertensive medication adherence; 203 (50.1%) were good weight management practitioners; 207 (51.1%) practiced a low-salt diet intake, 168 (41.5%) were good practitioners of the recommended level of physical activity; and 369 (91.1%) and 333 (82.2%) were nonsmokers and alcohol abstainers, respectively . Hypertensive patients with good self-efficacy had 3.33 times greater odds of practicing good self-care practice than those who had poor self-efficacy . The odds of having good self-care among patients with good knowledge of hypertension self-care practice were 6.36 times higher than those with poor knowledge of hypertension self-care practice . Moreover, patients without depression had 6.08 greater odds of having good hypertension self-care practices than patients with depression   of patients practiced good self-care. Based on the findings of this study around one in three hypertensive patients practiced good self-care practice as advised by the hypertension management protocol. This finding is in line with studies conducted in Harar (29.9%) , and WesThis study showed that the odds of having good self-care practice were 3.77 times higher among younger adults  compared with older adults . This result is supported by studies conducted in India , MekelleThis study indicated that the odds of practicing good self-care were 3.33 times greater than those with good self-efficacy compared with those who had poor self-efficacy. This result is similar to the findings of a systematic review that reported higher self-efficacy being associated with engagement in self-care behaviors . StudiesDepression was another significant factor associated with good self-care practices. In this study, patients without depression had sixfold greater odds of having good hypertension self-care practices than patients with depression. This finding is consistent with a study conducted in Deagu, Korea . This miThis study ascertained that patients with good knowledge of hypertension self-care practice were 6.36 times more likely to practice good self-care than those with poor knowledge. This finding is in agreement with studies done in Saudi Arabia , Iran 5 Addis AbSince the study participants' self-care practices have relied on self-reports, there may be a recall and social desirability bias.The study found that the level of good hypertension self-care practice in the study area was low. Age under 65 years, good knowledge of hypertension self-care practice, absence of depression, and good self-efficacy were independent predictors of good self-care practice. Continuous monitoring and follow-up of patient's adherence to the self-management protocol are required, with a greater focus on identifying factors that may hamper patients' adherence to the hypertension self-care protocols. To accomplish this, policymakers should consider these aspects when developing public health initiatives on hypertension self-care and when bolstering ongoing non-communicable disease reduction programs. Moreover, public health facilities should increase their efforts to give patients and families specialized instruction on all components of self-care practice.


Post hoc tests indicated significant performance improvements for the CCT-2 and CCT3 groups for all dependent variables , including the 15 and 30 m linear sprint speeds (p < 0.001), medicine ball throw (p < 0.001), standing long jump (p < 0.001), countermovement jump with arm thrust (p < 0.001), right leg knee extensor (p < 0.001) and flexor peak torque (p < 0.001), left leg knee extensor (p < 0.001) and flexor peak torque (p < 0.001), and change-of-direction speed (p < 0.001). The CCT-3 group showed greater improvements in MAT compared to the CCT-2 group . In conclusion, compared to active controls, the load-equated CCT-2 and CCT-3 programs provided similar effects on measures of physical fitness in active adult males. However, an athlete’s goal is to improve their MAT score, the CCT-3 program may elicit greater improvements compared with the CCT-2 program.Complex contrast training (CCT) is an exercise modality that utilizes both high-load resistance activity and low-load plyometric activity in a set-by-set fashion within a single exercise session. Such a combination of exercises targets multiple aspects of the force–velocity curve and may thus lead to improvement of various components of physical fitness. However, no previous study has attempted to compare the effects of load-equated two vs. three CCT sessions per week on measures of physical fitness. Forty-five male participants aged 21.4 ± 2.0 years were randomly assigned to either two weekly CCT sessions , three weekly CCT sessions , or an active control group . Selected measures of physical fitness were assessed pre- and post-six weeks of training. The tests included the assessment of 15 and 30 m linear sprint speeds, upper  and lower limb muscle power (standing long jump and countermovement jump with arm thrust), muscle strength (isokinetic peak knee extensor/flexor torque), and change-of-direction speed (modified agility T-test (MAT)). Significant group–time interactions were observed for all dependent variables (all  Sufficient levels of physical fitness  are important prerequisites for the performance of sports-specific motor skills and for the promotion of motor skill learning ,2. AlthoWe note that the combination of heavy load resistance exercises with plyometric exercises within a single training session may induce additional improvements in physical fitness that may further enhance physical fitness . The comFurthermore, with CCT, the exercise programming variables such as intensity  and total load  are well-researched . HoweverOf note, resistance training, plyometric jump training, and CCT may target the force–velocity continuum differently . Therefore, extrapolations from single-mode resistance training or plyometric jump training to CCT may be inappropriate. Accordingly, well-designed studies  that compare the effects of different weekly CCT frequencies are required. Therefore, this study aimed to contrast the effects of two vs. three load-equated weekly CCT training sessions on selected measures of physical fitness in active adult males, including 15 and 30 m linear sprint speeds, upper ) and lower limbs muscle power (standing long jump (SLJ), countermovement jump with arm thrust (CMJA)), muscle strength (isokinetic knee flexor/extensor peak torque), and change-of-direction speed (modified agility T-test (MAT)). Based on the available literature ,25, we hThe study was designed according to the international guidelines for quality-based randomized controlled trials ,29,30. −1, respectively, during the baseline and post-intervention assessments. A two  by three  randomized study design was used to compare the effects of different exercise frequencies on linear sprint speed, muscle power and strength, and change-of-direction speed. Pre- and post-intervention measurements were performed at similar times during the day for all participants, with linear sprints, MBT, SLJ, CMJA, and MAT conducted on day one (6:00–8:00 AM) and isokinetic testing conducted 24–72 h after day one (2:30–5:30 PM). The sequence of the testing order was the same for all participants and tests (pre- and post-intervention). Upon arrival in the laboratory, the participants underwent a 10-min general warm-up. For outdoor assessments, temperature, humidity, and wind velocity ranged from 28–31 °C, 15–65 %, and 0–10.8 km.hParticipants performed three familiarization sessions including CCT exercises and two familiarization sessions for the testing procedures that were undertaken one and two weeks before the baseline testing. Demographic and anthropometric data were collected and 1-RM tests were performed at least one week before the baseline testing during the familiarization sessions, and these included squats, barbell lunges, Romanian deadlifts, and bench presses. The results from the 1-RM testing were used to program the exercise interventions. The participants were asked to (i) refrain from strenuous activity 24 h before testing, (ii) eat and drink habitually, and (iii) refrain from consuming caffeine three hours before testing. A schematic representation of the study is provided in The required sample size for this study was estimated using statistical software . The following variables were included in the a priori power analysis: study design with three groups; two measurements; an alpha error of <0.05; a non-sphericity correction of 1; a correlation between repeated measures of 0.5; a desired power (1-ß error) of 0.80; and an effect size (f) of 0.27, all based on prior research investigating the effects of six-weeks of CCT on amateur soccer players’ 30 m linear sprint speed performance .www.randomizer.org) to either two weekly CCT sessions , three weekly sessions , or an active control group . The number of total repetitions and load used in the experimental groups was equated across the groups. The participants within each group possessed similar demographics and anthropometrics, except for body mass (p = 0.028) ). Accordingly, 45 male participants were recruited for this study, with a slightly higher number of participants than recommended in case any participants dropped out . The eligibility criteria for this study required participants who were: (1) university students, (2) actively participating in sports  as part of their course curriculum or in other forms of physical activity  for a minimum duration of five hour per week, (3) having a minimum of one year of resistance training/testing experience and able to perform all the exercises included in the study’s training protocol, and (4) free from lower limb injuries for at least six months before this study. Participants were randomly assigned (using the randomization tool = 0.028) . The potBefore the start of the training intervention, 1-RM assessments were conducted according to the methods outlined in a previous study . Before Six weeks of CCT interventions were considered , and bioAll the measurements were conducted by the same independent assessors pre- and post-intervention. In addition, the assessors were blinded to the group allocation of the participants.Linear sprint speed protocols were adapted from the methods outlined in a previous study  and condFor the performance of the MBT, participants stood on the start line with their feet shoulder-width apart. Thereafter, participants threw a three kg medicine ball backward overhead. The distance between the start line and where the ball first contacted the floor was measured using a standard measuring tape. The test was performed as described previously by Stockbrugger and Haennel . Two triThe SLJ protocol was adapted from methods outlined in a previous study  and condp = 0.550)) for measuring the CMJ performance. The sensor was placed on the lower back using a belt with the center of the device at the fifth lumbar vertebrae. Participants stood with their feet slightly apart and were instructed to swing their arms and perform a countermovement to a self-selected depth before taking off and landing with both legs. Knee flexion was not permitted during the flight phase of the jump. Three trials were performed with one minute of rest between jumps, and the best trial was selected for analysis. The ICC for test–retest was 0.96 (95% CI: 0.93–0.98).An inertial moment sensor  was used to measure the countermovement jump performance with arm thrust (CMJA). A pilot study reported the sensor to be valid and reliable . A 10-min warm-up was completed before the test, and it included jogging and dynamic stretching of the lower limbs. Thereafter, the participants sat on the machine’s chair, with the axis of rotation of the dynamometer arm aligned with the axis of rotation of the knee. The ‘Knee Extension/Flexion’ test was selected to be performed with the isokinetic ‘CONC/CONC’ mode; therefore, all knee extension and flexion movements involved concentric actions. The right side was always selected first across all testing sessions. The test protocol included a set of six repetitions at 60°/seconds. Two sets were completed for each leg with one minute of rest between the sets. Verbal instructions were provided to push and pull as hard and fast as possible throughout the full range of motion. Furthermore, the screen was positioned so that participants could see the real-time feedback on their effort. Two sets were performed, and the highest peak torque value obtained was selected for analysis. The ICC for test–retest was 0.99 (95% CI: 0.98–0.99) for right knee extension, 0.98 (95% CI: 0.97–0.99) for right knee flexion, 0.99 (95% CI: 0.98–0.99) for left knee extension, and 0.98 (95% CI: 0.97–0.99) for left knee flexion.post − meanpre)/meanpre) × 100. Effects sizes (ES) in the form of partial eta squared (ɳp2) were used from the ANOVA output. A Hedge’s g derived from the paired t-test was calculated to assess the group-specific changes between the pre- and post-intervention measurements. The magnitude of effects for the ɳp2 was interpreted as small (<0.06), moderate (≥0.06–0.13), and large (≥0.14) [g was interpreted as trivial (<0.2), small (0.2–0.6), moderate (>0.6–1.2), large (>1.2–2.0), very large (>2.0–4.0), and extremely large (>4.0) [95%CI lower bound) [p ≤ 0.05.Following an intention-to-treat approach, the analyses were conducted using IBM SPSS version 20.0.0 . Data normality was tested and confirmed using the Shapiro–Wilk test. Accordingly, the data are presented as means and standard deviations. A two (time: pre- and post-intervention tests) by three  mixed ANOVA for repeated measures was used to analyze the exercise-specific effects. Furthermore, in case of significant group–time interactions, Bonferroni-adjusted paired (within-group) and independent (between-group comparisons at post-intervention) t-tests were used for the post hoc analyses. In the case of significant between-group baseline differences, an ANCOVA with baseline values as covariates was further used to verify the intervention effects. Bonferroni-adjusted post hoc tests were applied. Percentage change scores were calculated for each variable in each group using the following equation in Microsoft excel: ((mean (≥0.14) , while te (>4.0) . The ICCr bound) . Statistp = 0.061–0.864) were observed for the measures of linear sprint speeds, SLJ, and isokinetic peak knee flexor/extensor torque. However, significant between-group (CCT-3 vs. CCT-2) baseline differences were found for the upper/lower limb muscle power (MBT and CMJA) and change-of-direction speed (MAT), with significantly lower values in the CCT-3 group (p = <0.001–0.01). All participants received the treatments as allocated. No training- or test-related injuries were observed. Adherence to training was 100%. The results for all dependent variables of the main analysis are presented in p < 0.001, ɳp2 = 0.51–0.78), with post hoc tests revealing differences in all variables that favored the CCT-2 and CCT-3 groups compared to the CG group . The MATs at the post- intervention tests favored (p < 0.001) the CCT-3 group (g = 3.26) compared to the CCT-2 group (g = 0.70). Within-group analyses revealed improvements in all dependent variables in the CCT-2  and CCT-3 groups , but not in the CG group .Significant group–time interactions were observed for all dependent variables   and strut-test p = < 0.001), with the CCT-3 group being comparatively slower than the CCT-2 group at baseline (mean: 6.39 s vs. 5.84 s). Therefore, the CCT-3 group had a greater opportunity to improve during the MAT. Furthermore, greater training frequency may have also allowed for greater neuromuscular adaptations and improved running efficiency . However, for the 15 m sprint, the baseline scores were not different. Therefore, the improvement in the 15 m sprint may be solely attributed to the greater training frequency, which may have led to adaptations that enhanced the utilization of the stretch–shortening cycle. Indeed, previous studies have suggested that more frequent neuromuscular stimuli may optimize motor learning [Of note, we observed a slightly higher magnitude of improvement in the 15 m sprint (g = 1.35 vs. 0.97) and MAT (g = 3.26 vs. 0.70) for the CCT-3 group compared to the CCT-2 group, and in the SLJ (g = 1.12 vs. 0.63) and CMJA (1.61 vs. 1.06) for the CCT-2 group compared to the CCT-3 group. The greater improvements observed in MAT performance can be partially explained by the window of adaptation mechanism . The baslearning , and thuAdditionally, the baseline score for CMJA was significantly greater for the CCT-2 group compared to the CCT-3 group (mean: 44.6 cm vs. 38.03 cm). Contradictory to previous findings observed for the MAT, here, we observed that the group (CCT-2) with a greater CMJA performance at baseline  had a larger magnitude of improvement. The lower training frequency may have also allowed for greater recovery, thereby improving jump performance . However, the interpretation of results such as greater improvements in the CCT-2 group for jumps compared to greater improvements in the CCT-3 group for the l5 m sprint and MAT may be possible through electromyographic analysis during the activities. Additionally, with similar lower limb maximal strength (1-RM for squat and Romanian deadlift), a CCT program appears to be more effective in improving vertical jumps for individuals with greater explosiveness. Whether such a mechanism exists needs to be confirmed in future studies with post-activation performance enhancement assessments across individuals with similar maximal strength, but with different explosive power capabilities. Furthermore, no differences in improvements were observed between both CCT training frequencies for the30 m linear sprint, MBT, and isokinetic leg strength. Results for the isokinetic leg strength test indicated that there were no differences in the magnitude of improvement between the CCT-2 and CCT-3 groups, which is in line with previous literature that has suggested that training frequency is a less decisive moderator for lower limb strength when training load is equated ,43,44. Although the findings derived from this study may be useful for practitioners for making evidence-based decisions regarding weekly CCT frequency to optimize physical fitness improvements, potential limitations of this study should be considered when interpreting our findings. Firstly, the participants included in the study were only active male students. Therefore, the extrapolation of the findings from this study to other populations  should be made with caution. Secondly, the study was limited to a six-week duration. Although training adaptations tend to occur after six weeks, a study of longer duration should be conducted to verify the long-term effects of load-equated frequency moderators. Thirdly, although a sample size estimation was conducted, a larger sample size may be required for more robust conclusions. Fourthly, the absence of biomechanical or physiological comparisons in this study limited a more comprehensive interpretation and explanation of results. Fifthly, although we found excellent inter-assessor reliability for both the sprint and MAT measurement protocols using a stopwatch, the inclusion of electronic timing gates would be recommended in future studies to further improve precision. Lastly, the inclusion of subjective measures of effort or fatigue such as the session rating of perceived exertion or the readiness questionnaire would have offered an additional insight into the understanding of physical fitness and the related psychobiological responses to different CCT frequencies.Compared to the active controls, the load-equated CCT-2 and CCT-3 programs appear to have had similar effects on most measures of physical fitness in active adult males. Of note, the CCT-3 program provided greater improvements compared to those of the CCT-2 program for the change-of-direction speed. Therefore, practitioners can use this information as a basis for designing training programs to elicit the desired physical fitness adaptations in their athletes. Moreover, for active adults with no previous CCT experience, a progressive load increment of 10% of the individual’s 1-RM every fortnight, with lower loads  during the initial weeks for primarily anatomical adaptations. Furthermore, we did not monitor any training or test-related injuries in this study; therefore, it appears that the implementation of CCT was safe and effective for both training frequencies.